Figure 4

Depletion of REEP5 and REEP6 causes a defect in IL-8-stimulated internalization of CXCR1 and recruitment of β-arrestin2.
(A) HEK293 cells containing shRNAs were transfected with CXCR1 and β-arrrestin2-GFP plasmids, treated with 10 nM IL-8 for 30 min, and fixed with 4% PFA. GFP signals were detected by confocal microscopy. (B) The bar graph presents the percentage of cells showing different internalization signals. The intensity was arbitrarily determined by brightness. The graph describes % of total cells. Supplementary Fig. S3 shows clustering patterns of β-arrestin2-GFP determined as strong, mild, and none. One way-ANOVA was performed between control group(sc) and REEP-knockdown cells(sh) for each clustering pattern of β-arrestin2-GFP. **p < 0.01. (C) IL-8-stimulated β-arrestin2 internalization in cells expressing CXCR2 was not affected by the depletion of REEP5 and REEP6. (D) HEK293 cells containing shRNAs were transfected with the CXCR1-GFP alone or CXCR1-GFP and REEP5-His plasmids. The cells were treated IL-8 for 30 min and applied for immunostaining with anti-His antibodies. GFP signals were detected by confocal microscopy. All images are representative of three independent experiments. NT, not treated; sc, scrambled shRNA; sh, shRNA. The scale bar denotes 10 μm. All experiments were performed at least three times.